Voltage gated K+ (Kv) channels couple the flux of K+ to the membrane potential and play key roles in the brain and heart. Mutations in Kv channels can cause severe diseases in humans such as epilepsies and cardiac arrhythmias. There have been major advances in the structure determination of Kv channels. In spite of the structural information available, there are major questions on the functional mechanisms in Kv channels that remain unanswered. Here we investigate the processes of voltage gating and C-type inactivation that regulate the flux of K+ through Kv channels. We use a multidisciplinary approach centered on unnatural amino acid (UAA) mutagenesis in our investigations. UAA mutagenesis is a very powerful method for protein modification, compared to traditional mutagenesis, because it allows a large variety of side chain modifications and also permits the modification of the protein backbone. We use this approach to investigate the role of the main chain H-bonds in the fourth transmembrane helix (TM4) in voltage gating of the Shaker K+ channel and the hyperpolarization activated and cyclic nucleotide gated ion channel HCN (aim 1). We investigate the role of ion binding sites in the selectivity filter of the Shaker channel in C-type inactivation and we complement the functional studies on Shaker with structural studies on the KvAP channel, an archaeal homolog of the Shaker channel (aim 2). We also investigate the mechanism of C-type inactivation in the hERG K+ channel, which has interesting functional differences from C-type inactivation in the Shaker channel and is physiologically critical for normal cardiac function (Aim 3). The research proposed is significant as it provides greater insight into the functional mechanisms of voltage gating and C-type inactivation in Kv channels. The research is also significant as it will provide a general strategy for using UAA mutagenesis to investigate the role of main chain H-bonds and ion binding sites, which are important for function in many families of membrane proteins.